Despite advances of modern medicine, bacterial sepsis remains one of the major threats to human health worldwide. In the U.S.A alone, it affects approximately 750,000 Americans per year, with an associated mortality rate >28%. The lipopolysaccharide (LPS), an outer membrane component of Gram negative bacteria, and various structures of Gram positive bacteria and Mycobacterium tuberculosis, represent the initiating stimuli in the activation of the innate immune response. Effective recognition of these bacterial structures by Toll-like receptors (TLR) expressed on monocytes and macrophages is critical for mounting a strong first line defense and prevention of sepsis. Many patients with sepsis develop a hypoinflammatory state that is manifested by marked inhibition of monocyte functions, including suppressed production of a number of cytokines. This state is highly reminiscent of bacterial tolerance defined as a transient state of cell refractoriness following a prior exposure to bacterial components. Therefore, induction of bacterial tolerance can be used as a model to delineate mechanisms that underlie decreased monocyte responsiveness in patients with sepsis. Despite numerous studies, the molecular mechanisms that underlie induction of bacterial tolerance are still largely unknown. The overall goal of this project is to gain a better understanding of the molecular mechanisms of host responses to microbes. Our objective is to define the role of receptors and signal transducing molecules of the TLR pathway in induction and maintenance of bacterial tolerance in monocytes. The central hypothesis is that induction of bacterial tolerance in human monocytes dramatically alters signal-induced complex formation among key components of the TLR pathway, leading to development of a state of suppressed antibacterial responsiveness. The following Specific Aims are proposed to: 1. Examine post-translational modifications and interactions of key molecules of TLR4 complexes associated with tolerance to TLR4 and TLR2 agonists. 2. Analyze the effect of bacterial tolerance on expression, recruitment, post-translational modifications of key adapter proteins and activation of IRAK-4. 3. Characterize suppressors of TLR-mediated signaling in tolerized cells that interfere with agonist-induced interactions among TLRs, adapter proteins, and IRAK kinases. It is expected that at the completion of this grant, we will have identified key molecular mechanisms responsible for development of bacterial tolerance, and have provided strategies for development of new therapeutic approaches for treatment of patients with bacterial sepsis to target components of the TLR signaling pathway that are compromised in tolerance and sepsis.